The soluble cytokine erythropoietin (Epo) plays a central role in physiologic erythropoiesis and is a mainstay in treatment of human anemias. Two major problems associated with Epo administration are treatment resistance and adverse side-effects. Treatment resistance usually occurs at the level of the target cells, the erythroid progenitors. The most prominent adverse side-effects of Epo consist of cardiovascular complications, particularly congestive heart failure (CHF). Although Epo signaling has been studied for several decades, the mechanistic basis for these important clinical problems remains unknown. Furthermore, the basic question of how Epo signaling promotes erythroid differentiation remains itself unresolved. It has been known for over a decade that Epo somehow regulates the function of the master regulator of erythroid gene transcription, GATA-1. Activation of GATA-1 in the absence of Epo signaling causes cell death without differentiation. Although several models have been proposed for Epo regulation of GATA-1, no mechanism has been established. This proposal provides evidence for Epo regulation of GATA-1 through a protein kinase D/class IIa histone deacetylase (PKD/HDAC) pathway, building on previous observations of GATA-1 binding and repression by class IIa HDACs. This evidence, obtained in both G1ER and human erythroid progenitors, includes the activation of PKD by Epo and the blockade of Epo-driven erythroid differentiation through PKD inhibition. Further evidence consists of the enhancement of erythroid Epo responsiveness through HDAC5 knockdown and elimination of GATA-1 dependence on Epo signaling by an HDAC inhibitor. Importantly, class IIa HDAC signaling pathways have been implicated in the pathophysiology of cardiac hypertrophy and CHF, wherein class IIa HDAC phosphorylation by PKD or CaMK causes inappropriate activation of the myogenic transcription factor MEF2. Thus Epo activation of PKD may promote both GATA-1-driven erythropoiesis and MEF2-driven CHF. In this proposal, we will determine the mechanism for Epo activation of PKD, examining the structural requirements of the EpoR and its coregulator, c-Kit. In addition, the essential EpoR downstream mediators, JAK2 and PLC3, will be analyzed for their contributions to PKD activation. We will also examine whether Epo adminstration induces PKD activation in vivo in myocardium and, if so, which EpoR-associated elements contribute to this signaling. These experiments will thus delineate the upstream components of a novel Epo signaling pathway involved in programming of erythropoiesis and potentially in induction of CHF. The information gained from these studies will establish a conceptual framework for probing mechanisms of clinical Epo resistance and for design of pharmacologic agents to reverse Epo resistance. Of equal importance, these experiments will mechanistically address the unresolved issue of Epo's association with cardiovascular complications. This proposal is responsive to the program announcement Stimulating Hematology Investigation: New Endeavors (PAS-10-046). One specific topic listed in this announcement is Non-erythroid Expression and Function of Erythropoietin Receptors. A significant component of this project will be devoted to analysis of myocardial PKD activation by Epo administration in mice with wild type and mutant EpoR, as well as in mice with wild type and mutant c-Kit. Another topic listed is Anemia of Inflammation and Chronic Disease. Improved understanding of normal erythroid EpoR signaling is a critical first step toward understanding the mechanisms of Epo resistance associated with these anemias. PUBLIC HEALTH RELEVANCE: Erythropoietin (Epo) is critical for normal red cell production and is administered as a treatment to numerous patients with anemia. A published, large-scale clinical trial has shown that giving patients sufficient doses of Epo to completely correct anemia, rather than partially correct anemia, confers an increased risk of cardiovascular disease, particularly congestive heart failure (CHF). Despite the tremendous amount of resources invested in Epo therapy and research, relatively little is understood about how it may promote red cell production as well as heart disease. In this proposal, a new signaling pathway, that can potentially explain both the beneficial and deleterious effects of Epo, will be characterized.